U.S. Pat. No. 3,139,428 teaches compounds of formula ##STR2##
wherein R.sub.1 is hydrogen, methyl, or R.sub.3 ; R.sub.2 is hydrogen, keto, hydroxyl or a radical of the formula --OR.sub.4 in which R.sub.4 is the acyl radical of an aliphatic carboxylic acid containing 2 to 6 carbon atoms such as acetyl, propionyl, butyryl, valeryl, hexanoyl and the like, benzoyl or benzoyl substituted with 1, 2, or 3 lower alkyl, halo or lower alkoxy groups, for example p-methylbenzoyl, m-methylbenzoyl, o-methylbenzoyl, 3,4-dimethylbenzoyl, p-chlorobenzoyl, p-fluorobenzoyl, o-bromobenzoyl, m-chlorobenzoyl, 2,4-dichlorobenzoyl, p-methoxybenzoyl, 3,4,5-trimethoxybenzoyl, 3,4-dimethoxybenzoyl and the like; and R.sub.3 is .dbd.CHR.sub.5 or --CH.sub.2 R.sub.6 in which R.sub.5 is lower alkyl, furyl, phenyl, phenyl lower alkyl such as benzyl, phenethyl, phenylpropyl and the like, phenyl lower alkenyl such as cinnamyl, phenylpropenyl, phenylbutenyl and the like, methylenedioxyphenyl, or phenyl nuclearly substituted with 1 or 2 halo, lower alkyl, lower alkoxy or nitro groups such as p-chlorophenyl, 3,4-dichlorophenyl, m-methylphenyl, o-bromophenyl, p-ethylphenyl, p-fluorophenyl, p-methoxyphenyl, o-methoxyphenyl, m-butoxyphenyl, p-nitrophenyl, 3,4-dinitrophenyl, 2,4-dinitrophenyl, o-nitrophenyl, m-nitrophenyl and the like, and R.sup.6 is lower alkyl, furyl, phenyl, phenyl lower alkyl, methylenedioxyphenyl, or phenyl nuclearly substituted with 1 or 2 halo, lower alkyl, lower alkoxy or amino groups. The compounds are disclosed as useful as intermediates and as anti-inflammatory agents.
U.S. Pat. No. 3,291,800 teaches compounds of formula ##STR3##
wherein R.sub.1 represents lower alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl and the like, aryl such as phenyl or aralkyl such as benzyl; R.sub.2 represents hydrogen, lower alkyl such as methyl, ethyl, propyl, isopropyl and the like, lower alkoxy such as methoxy or ethoxy or acetyl; R.sub.3 represents hydrogen or lower alkyl and R.sub.4 represents hydrogen hydroxy or keto and to the nontoxic pharmaceutically acceptable acid addition and quaternary ammonium salts thereof. The compounds are disclosed as having pharmacological activity as analgesics, tranquilizers, and anti-inflammatory agents.
These patents are hereby incorporated by reference.
Migration of leukocytes from blood vessels into diseased tissues is important to the initiation of normal disease-fighting inflammatory responses. But this process, known as leukocyte recruitment, is also involved in the onset and progression of debilitating and life-threatening inflammatory and autoimmune diseases. The pathology of these diseases results from the attack of the body's immune system defenses on normal tissues. Thus, blocking leukocyte recruitment to target tissues in inflammatory and autoimmune disease would be a highly effective therapeutic intervention. The leukocyte cell classes that participate in cellular immune responses include lymphocytes, monocytes, neutrophils, eosinophils and basophils. In many cases, lymphocytes are the leukocyte class that initiates, coordinates, and maintains chronic inflammatory responses, and thus are generally the most important class of cells to block from entering inflammatory sites. Lymphocytes attract monocytes to the site, which, collectively with lymphocytes, are responsible for much of the actual tissue damage that occurs in inflammatory disease. Infiltration of lymphocytes and/or monocytes is responsible for a wide range of chronic, autoimmune diseases, and also organ transplant rejection. These diseases include, but are not limited to, rheumatoid arthritis, atherosclerosis, psoriasis, chronic contact dermatitis, inflammatory bowel disease, multiple sclerosis, sarcoidosis, idiopathic pulmonary fibrosis, dermatomyositis, skin pemphigoid and related diseases, (e.g., pemphigus vulgaris, p. foliacious, p. erythematosis), glomerulonephritides, vasculitides, hepatitis, diabetes, allograft rejection, and graft-versus-host disease.
This process, by which leukocytes leave the bloodstream and accumulate at inflammatory sites, and initiate disease, takes place in at least three distinct steps which have been described as (1) rolling, (2) activation/firm adhesion and (3) transendothelial migration (Springer T. A., Nature 1990;346:425-433; Lawrence and Springer, Cell 1991;65:859-873; Butcher E. C., Cell 1991;67:1033-1036). The second step is mediated at a molecular level by chemoattractant receptors. Chemoattractant receptors on the surface of leukocytes bind chemoattractant cytokines secreted by cells at the site of damage or infection. Receptor binding activates leukocytes, increases the adhesiveness of the adhesion molecules that mediate transendothelial migration, and promotes directed migration of the cells toward the source of the chemoattractant cytokine.
A recent discovery is the existence of a large family (&gt;20 members) of structurally homologous chemoattractant cytokines, approximately 8 to 10 kD in size. These molecules share the ability to stimulate directed cell migration (chemotaxis) and have been collectively called "chemokines," a contraction of chemotactic cytokines. Each chemokine contains four cysteine residues (C) and two internal disulfide bonds. Chemokines can be grouped into two subfamilies, based on whether the two amino terminal cysteine residues are immediately adjacent (C--C family) or separated by one amino acid (C--X--C family). These differences correlate with the organization of the two subfamilies into separate gene clusters. Within each gene cluster, the chemokines typically show sequence similarities between 25% to 60%.
The chemokines of the C--X--C subfamily, such as interleukin-8, are produced by a wide range of cell types and act predominantly on neutrophils as mediators of acute inflammation. Chemokines of the C--C subfamily are also produced by a wide variety of cell types. These molecules act predominantly on subsets of mononuclear inflammatory cells. Currently there are at least six C--C chemokines with known chemotactic activity for human monocytes and/or T cells, including MCP-1, MCP-2, MCP-3, MIP-1.alpha., MIP-1.beta., and RANTES. This suggests there may be a high degree of redundancy in chemoattractant pathways. In addition, most C--C chemokines are chemotactic for more than one cell type. For examples, RANTES (regulated on activation, normal T cell expressed and secreted) acts on memory CD4.sup.+ T cells, eosinophils, and monocytes. Monocyte chemoattractant protein-1 (MCP-1), another C--C chemokine, acts on monocytes, activated "memory" T cells and on basophils. MCP-1 is also a potent secretogogue of inflammatory mediators for monocytes and basophils.
Five C--C chemokine receptors have recently been characterized (CCRR1-5 or CCR1-CCR5), and all of these belong to the seven transmembrane spanning G protein-coupled receptor family. Each of these receptors mediates the binding and signaling of more than one chemokine. For example, the CCR1 receptor binds both MIP-1.alpha. and RANTES. There are 2 receptors which bind MCP-1, CCR2 (with alternately spliced forms, 2A and 2B) and CCR4. CCR2 is also known to mediate binding and signaling of MCP-3. The CCR4 receptor binds and signals, in addition to MCP-1, with RANTES and MIP-1.alpha.. Which of these is responsible for the MCP-1 mediated recruitment of monocytes and T cells is not known.
In agreement with the observation that lymphocyte emigration into inflammatory sites is usually accompanied by emigration of monocytes, MCP-1 is expressed at sites of antigen challenge and autoimmune disease. However, analyses of human inflammatory lesions with antibodies to other chemokines show RANTES, MIP-1I, MIP-1.theta. and MCP-3 to be present as well. Injection of MCP-1 into skin sites in mice provokes only a mild monocytic infiltrate or no infiltrate at all (Ernst C. A. et al., J. Immunol. 1994;152:3541-3544). Whether these results reflect redundant and complex recruitment pathways has not been resolved. MCP-1 and MCP-3 may play a role in allergic hypersensitivity disease. This is suggested by the observation that MCP-1 lacking the amino terminal glutamic acid loses the ability to stimulate basophil mediator release and acquires activity as an eosinophil chemoattractant.
Chemokines of both subfamilies may bind to heparan sulfate proteoglycans on the endothelial cell surface, and may function principally to stimulate haptotaxis of leukocytes that attach to cytokine-activated endothelium through induced adhesion molecules. Additionally, MCP-1 has been reported to selectively activate the .beta.1 integrin family of leukocyte adhesion molecule, suggesting a role in leukocyte interactions with the extracellular matrix. Hence, MCP-1 may not only trigger the initial arrest and adhesion of monocytes and T cells, but may also act to guide their migration in extravascular space.
Chemoattractants appear to be required for transendothelial migration in vitro and in vivo and can induce all steps required for transmigration in vivo. Injection of neutrophil chemoattractants into skin or muscle leads to robust emigration of neutrophils from the vasculature and accumulation at the injection site (Colditz, 1991). Pretreatment of neutrophils with pertussis toxin inhibits emigration into inflammatory sites (Spangrude et al., 1985; Nourshargh and Williams, 1990). Moreover, MAb to IL-8 markedly inhibits neutrophil emigration in inflammation (Sekido et al., 1993).
Neutrophil chemoattractants injected into the same skin site hours apart will stimulate neutrophil accumulation the first time but not the second time, whereas a second injection into a distant site will stimulate accumulation at that site. This desensitization occurs for homologous chemoattractants only (Colditz, 1991) or those that interact with the same receptor. Thus, chemoattractants can act on and homologously desensitize a cell type that is localized in tissue.
Chemokines mediate a range of proinflammatory effects on leukocytes, such as chemotaxis, degranulation, and intigran activation (Baggiolini et al., Adv. Immunol., 1994;55:97-179; Oppenheim et al., Annu. Rev. Immunol., 1991; 9:617-48; Miller et al., Crit. Rev. Immunol., 1992;12:17-46). These effects are mediated by binding to the seven-transmembrane-spanning G-protein coupled receptors (Baggiolini et al., Adv. Immunol., 1994;55:97-179; Murphy, Annu. Rev. Immunol., 1994;12:593-633; Schall et al., Curr. Opin. Immunol., 1994;6:865-73; Gerard et al., Curr. Opin. Immunol., 1994;6;140-5; Mackay, Curr. Bio., In press). Chemokine receptors also serve as coreceptors for HIV-1 entry into cells. This came from observations that RANTES, MIP-1.alpha., and MIP-1.beta. suppressed infection of susceptible cells in vitro by macrophage-tropic primary HIV-1 isolates (Cocchi et al., Science (Wash. D.C.), 1995;270:1811-5). The chemokine receptor CXCR-4 was found to support infection and cell fusion of CD4.sup.+ cells by laboratory-adapted, T-tropic HIV-1 strains (Feng et al., Science (Wash. D.C.), 1996;272:872-7). CCR-5, a RANTES, MIP-1.alpha., and MIP-1.beta. receptor, was subsequently identified as the principle coreceptor for primary macrophage-tropic strains (Choe et al., Cell, 1996;85: 1135-48; Alkhatib et al., Science (Wash. D.C.), 1996;272: 1955-8; Doranz et al., Cell, 1996;85: 1149-58; Deng et al., Nature (Lond.) 1996;381:661-6; Dragic et al., Nature (Lond.), 1996;381:667-3). The importance of CCR-5 for HIV-1 transmission was underscored by the observation that certain individuals who had been repeatedly exposed to HIV-1 but remained uninfected had a defect in CCR-5 expression (Liu et al., Cell, 1996; 86:367-77; Samson et al., Nature (Lond.), 1996;382:722-5; Dean et al., Science (Wash. D.C.), 1996;273:1856-62; Huang et al., Nature Med., 1996;2:1240-3). These noninfectable individuals were found to be homozygous for a defective CCR-5 allele that contains an internal 32-base pair deletion (CCR-5 .DELTA.32). The truncated protein encoded by this gene is apparently not expressed at the cell surface. CCR-5 .DELTA.32 homozygous individuals comprise .about.1% of the Caucasian population and heterozygous individuals comprise .about.20%. In studies of about 2700 HIV-1 infected individuals, no .DELTA.32 homozygotes were found. Individuals who are heterozygous for .DELTA.32 CCR-5 allele have been shown to progress more slowly to AIDS than wild-type homozygous individuals (Samson et al., Nature (Lond.), 1996;382:722-5; Dean et al., Science (Wash. D.C.), 1996;273:1856-62; Huang et al., Nature Med., 1996;2:1240-3). Thus, the identity of CCR-5 as the principle coreceptor for primary HIV isolates provides an opportunity to understand disease pathogenesis, and more importantly to identify a new avenue for the treatment of HIV-1 infection.
Chemoattractants impart directionality to leukocyte migration. By contrast with intradermal injection, intravascular injection of IL-8 does not lead to emigration (Hechtman et al., 1991). Cytokine-stimulated endothelial monolayers grown on filters secrete IL-8 into the underlying collagen layer. Neutrophils added to the apical compartment emigrate into the basilar compartment, but not when the IL-8 gradient is disrupted by addition of IL-8 to the apical compartment (Huber et al., 1991).
The endothelium may present chemoattractants to leukocytes in a functionally relevant way, as well as providing a permeability barrier that stabilizes the chemoattractant gradient. Since leukocytes, responding to specific antigen or inflammatory signals in tissue, may signal emigration of further leukocytes into the site, a chemoattractant was sought in material secreted by mitogen-stimulated mononuclear cells (Carr et al., 1994). Purification to homogeneity guided by a transendothelial lymphocyte chemotaxis assay revealed that monocyte chemoattractant protein 1 (MCP-1), previously thought to be solely a monocyte chemoattractant, is a major lymphocyte chemoattractant. An activated subset of memory lymphocytes respond to MCP-1. In the same assay, lymphocytes respond to RANTES and MIP-1.alpha. but less so than to MCP-1 (C--C chemokines) and not at all to IL-8 or IP-10 (C--X--C chemokines). This physiologically relevant assay suggests that C--C chemokines tend to attract both monocytes and lymphocytes. In agreement with the observation that lymphocyte emigration into inflammatory sites is accompanied by emigration of monocytes, MCP-1 is abundantly expressed at sites of antigen challenge and autoimmune disease (Miller and Krangel, 1992) and, together with other chemokines, is an excellent candidate to provide the step 2 signal required to activate integrin adhesiveness and emigration of lymphocytes in vivo. (Traffic Signals for Lymphocyte Recirculation and Leukocyte Emigration: The Multistep Paradigm; Springer, Cell 1994;76: 301-314).
We have surprisingly found the compounds of the invention are MCP-1 receptor antagonists and are capable of inhibiting the binding of MCP-1 to its receptor. Surprisingly, the compounds block T cell migration in vitro, and more surprisingly still, have dramatic effects on the recruitment of inflammatory cells in multiple models of inflammatory diseases. Thus, these compounds are useful as agents for the treatment of inflammatory disease, especially those associated with lymphocyte and/or monocyte accumulation, such as arthritis, atherosclerosis and transplant rejection. In addition, these compounds can be used in the treatment of allergic hypersensitivity disorders such as asthma and allergic rhinitis characterized by basophil activation and eosinophil recruitment, as well as for the treatment of restenosis and chronic or acute immune disorders.
The importance of CCR-5 for HIV-1 transmission was underscored by the observation that certain individuals who had been repeatedly exposed to HIV-1 but remained uninfected had a defect in CCR-5 expression (Liu et al., Cell, 1996; 86:367-77; Samson et al., Nature (Lond.), 1996;382:722-5; Dean et al., Science (Wash. D.C.), 1996;273:1856-62; Huang et al., Nature Med., 1996;2:1240-3). These noninfectable individuals were found to be homozygous for a defective CCR-5 allele that contains an internal 32-base pair deletion (CCR-5 .DELTA.32). The truncated protein encoded by this gene is apparently not expressed at the cell surface. CCR-5 .DELTA.32 homozygous individuals comprise .about.1% of the Caucasian population and heterozygous individuals comprise .about.20%. In studies of about 2700 HIV-1 infected individuals, no .DELTA.32 homozygotes were found. Individuals who are heterozygous for .DELTA.32 CCR-5 allele have been shown to progress more slowly to AIDS than wild-type homozygous individuals (Samson et al., Nature (Lond.), 1996;382:722-5; Dean et al., Science (Wash. D.C.), 1996;273:1856-62; Huang et al., Nature Med., 1996;2:1240-3). Thus, the identity of CCR-5 as the principle coreceptor for primary HIV isolates provides an opportunity to understand disease pathogenesis, and more importantly to identify a new avenue for the treatment of HIV-1 infection.